MXPA01004169A - Improved aav vector production. - Google Patents

Abstract

The invention relates to the field of genetically engineered viral vectors, more specifically to adeno-associated virus (AAV) vectors, for use in gene therapy. The present invention provides a process for the production of high titer recombinant adeno-associated virus vectors that are essentially free of helper virus such as adenovirus. The invention provides an adeno-associated virus (AAV) packaging cell having been provided with nucleic acid encoding a gene product providing AAV helper function allowing generating recombinant AAV without concomitant helper virus production.

Description

IMPROVED PRODUCTION OF ADENOSOCIATED VIRUS VECTOR DESCRIPTION OF THE INVENTION The invention relates to the field of genetically engineered viral vectors, more specifically with adeno-associated virus vectors (AAV), for use in gene therapy. Adeno-associated viruses are non-pathogenic human poroviruses (reviewed in (Berns, 1990a; Berns, 1990b)). Viruses reproduce as a single strand of DNA of approximately 4.6 kb. Both of the strands plus less are packaged and are infectious. The efficient reproduction of AAV requires coinfection of the cell by an auxiliary virus. Viruses that have been identified as AAV helper are adenoviruses, herpes simplex virus (HSV), cytomegalovirus (CMV) and pseudorabies virus (Berns, 1996). In the absence of an auxiliary virus, no substantial reproduction of the AAV is observed. The AAV is therefore also classified as a dependent virus. When an auxiliary virus is not present, the AAV genome can be integrated into the genome of the host cell. The natural virus has a strong preference (70%) for the integration site on the long arm of chromosome 19 (ql3.3-qter (Kotin et al, 1990, Samulski, 1993, Samulski et al, 1991). integration, the expression of the genes of the virus is not detectable, the integrated proviruses reproduce Itef: 12887B as a normal part of the genome of the host cell after the division of the transduced cells and ends in both daughter cells. This stage of the life cycle of the virus is known as the latent stage. This latent stage is stable but can be interrupted after infection of the cell transduced by an auxiliary virus. After infection of the helper virus, the AAV is excised from the genome of the host cell and begins to reproduce. During the initial phase of this Utico site, the rep genes are expressed. 10 Approximately 12 to 16 hours later capsule proteins VP1, VP2 and VP3 are produced in detectable quantities and the DNA of the reproduced virus is packaged in these virions. A schematic representation of the AAV genome and its genes are described in Figure 1. 15 Virions accumulate in the cell nucleus and are released when the cell is lysed as a result of accumulation of AAV and helper virus (reviewed in Berns, 1990a; Berns, 1990b). Thus, six primate AAV serotypes have been characterized (Berns et al, 1994, Rutledge 20 et al, 1998). The AAV genome contains two rep and cap genes (Fig. 1). Three promoters (P5, P19 and P40) direct the synthesis of mRNAs encoding 4 proteins Rep (Re? 78, Rep68, Rep52 and Rep40) and three capsule proteins (VPl, VP2 and VP3). The AAV genome is flanked on both sides by 25 a sequence of 145 bp, called the terminal repeat iÜtjM¡aaÉÉgMII_iÉtt _ * _ «_ * _ it? i"? i.- iiá-- .. .... i *. n -. . »» - ,. *? * Inverted (ITR), which seems to contain all the sequences that act in the cis position required for the integration, reproduction and encapsulation of the virus (Lusby et al, 1980; Samulski et al, 1989) . 5 During a productive infection the P5 promoter is activated first and directs the Rep78 and Repß8 production. These proteins are essential for AAV reproduction and trans regulation of viral genes. Rep52 and Rep40 are expressed from the P19 promoter and are thought to be 10 involved in the packaging of AAV genomes (Chejanovsky and Carter, 1989, Smith and Kotin, 1998). The VP1, VP2 and VP3 capsule proteins are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNA using the same 15 splice site but two different splice receiving sites. The splice receptor sites are located on both sides of the start signal of the VPl transduction. The VPl is translated from the messenger that uses the splice receiver directly on the front of the 20 codon of the VPl. VP2 and VP3 are translated from the messenger RNA that is spliced to the 3 'receptor of the VPl ATG. The proteins VP2 and VP3 are translated from this messenger but use a translation start of ACG (VP2) or downstream ATG (VP3). Since the three coding regions 25 are in box, capsule proteins share a -fftlhfT r - ?? r * - large domain with an identical amino acid sequence. VP3 is fully contained within VP1 and VP2, but the latter two contain additional amino-terminal sequences. Similarly, the VPl contains all the VP2 protein but contains an additional N-terminal sequence. The three proteins of the capsule end in the same position (Ruffing et al, 1994). The AAV capsule is 20 to 24 mm in diameter (Berns and Bohensky, 1987; Srivastava et al, 1983) and contains approximately 5% VPl, 5% VP2 and 10 90% of VP3. It is believed that this relationship reflects the relative abundance of the splice messengers, alternatively, and the efficiency of the start of translation reduced in the start codon of GCA for VP2. Adeno-associated virus vectors can 15 produced by replacing the rep and cap genes sequences in the natural AAV with the sequence of interest. To produce the recombinant virus, human cells infected with helper viruses need to be supplied concomitantly with the rep and cap genes through different means. This is done 20 routinely handle through the transfection of a so-called packaging plasmid, which provides the function for packaging AAV, which contains the AAV rep and cap genes but which lacks AAV ITR. The recombinant AAV is typically generated or by transducing a packaging plasmid 25 together with a plasmid containing the recombinant AAV in cells infected with helper virus. The recombinant virus is typically harvested from such cultures from 48 to 72 hours after transfection of the cells. The recombinant AAV generated in this way is of high titer and can be made essentially free of natural AAV (Alien et al, 1997, Samulski et al, 1989). Since cells are also concomitantly infected with an auxiliary virus, usually an adenovirus, this helper virus is also produced (Clark et al, 1997, Flotte et al, 1993, Herzog et al, 1997, Monahan et al, 1998, Snyder et al. al, 1997a). The reproduction of the AAV and also the packaging can be carried out in the test tube using a cell-free system (Hong et al, 1992, Hong et al, 1994, Ni et al, 1994, Zhou and Muzyczka, 1998, ard et al, 1998). The presence of helper virus in rAAV preparations is not desirable. Recombinant helper virus is a potential pathogen and even minor contaminations of recombinant AAV preparations with the helper virus are not acceptable for clinical use. Various methods are used to remove the helper virus from the recombinant AAV preparations. In the case of adenovirus those include differences in density and sensitivity to temperature. The AAV particles have a density of 1.41 to 1.45 g / cm3, while the adenovirus 2 and 5, the most commonly used helper viruses have a density of 1.33 g / cm3. With density gradient centrifugation this difference is used to separate the two viruses (Clark et al, 1997, Herzog et al., 1997). Differences in temperature sensitivity are also used to remove adenovirus 5 contaminants. The adeno-associated virus particles are more resistant to heat treatment than the adenovirus particles. Routinely, AAV preparations are incubated for 1 hour at 5 ° C. The recombinant AAV is resistant to this treatment, while the helper virus 10 adenoviruses (Flotte et al, 1993; Monahan et al, 1998; Snyder et al, 1997a). Although these methods are adequate to remove most auxiliary viruses, they are not ideal for clinical applications of recombinant AAV. One reason is that large clinical applications need to be produced 15 amounts of recombinant AAV. This implies that large amounts of helper virus must also be produced which must subsequently be completely removed from the rAAV preparation. In addition, the process of validating the absence of the auxiliary virus is difficult. The invention relates directly to the generation, production and purification of genetically engineered viral vectors designed to introduce and express a gene of interest in mammalian cells. The present invention provides a process for the production of 25 recombinant adeno-associated virus (AAV) vectors title, which are essentially free of helper viruses such as adenovirus. Several viruses can provide auxiliary functions for AAV. The auxiliary function of adenoviruses is currently the best characterized. In adenovirus, four regions have been identified that are required for a fully permissive AAV infection. These are the El, E2a, E4orf6 and VA regions. In the El, the genes of both the Ela and Elb regions are important. Studies in which the role of these genes has been discovered and characterized are reviewed in (Cárter, 1990). HSV can also function as a helper virus for AAV. HSV genes with identified helper virus function include ICP8 and ICP4 genes, viral DNA polymerase and possibly viral helicase (Berns, 1996). The invention provides a cell packaging adeno-associated viruses (AAV) that have been provided with nucleic acid encoding a genetic product that provides ancillary function for AAV, which allows generating recombinant AAV without concomitant helper virus production. More specifically, the invention provides methods, cell lines, recombinant adenoviral vectors and recombinant molecules especially suitable for the large-scale production of high titre recombinant AAV standards that are free of reproduction of competent adenoviruses. In normal cells the reproduction and packaging of AAV is undetectable. However, low level reproduction and packaging can be induced in the absence of helper virus function. Several methods have been published to induce a cycle of productive reproduction of AAV at a low scale. These include but are not limited to the treatment of cells with cytostatic drugs or UV irradiation (Yacobson et al, 1989, Yalkinoglu et al, 1988) and are not suitable for the large-scale production of recombinant high-grade AAV standards that are free of reproduction of competent auxiliary viruses. The invention provides a structurally better solution to completely avoid the generation of helper viruses during the production of recombinant AAV. The prevention of the generation of the auxiliary virus avoids the requirement of laborious purification and validation and subsequent testing of the preparations. The invention provides a way to eliminate the generation of helper viruses during the production of AAV by eliminating the requirement of helper virus for the reproduction of AAV. Methods to improve the production of recombinant AAV have attracted much attention in recent years. Several ways of expressing AAV rep and cap gens have been found to improve the performance of recombinant AAV over standard methods (Alien et al., 1997; Conway et al., 1997; Li et al., 1997; Vincent et al., 1997; ). In addition, the helper virus function has been under study and methods have been found to improve both the quality and the yield of the recombinant AAV preparations (Ferrari et al, 1997, Ferrari et al, 1996, Xiao et al, 1998b). In one aspect of the invention there is provided a packaging cell which expresses the E2A gene of adenovirus and additionally required auxiliary functions, where additionally required auxiliary functions do not possess sequence overlap with the auxiliary E2A function already present in the packaging cell, which lead to the formation of RCA. Preferably, the E2A gene is derived from the ts25 adenovirus. In another aspect of the invention there is provided a packaging cell which expresses the El region of adenovirus and the auxiliary functions additionally required, where the auxiliary functions additionally required do not possess sequence overlap with the auxiliary function of El already present in the packaging cell, that leads to the formation of RCA. In a particular aspect of the invention, the packaging cell comprises PER cell lines. The PER cell lines have been generated from normal human embryonic retinoblastic cells (HER) which were immortalized with a fully characterized plasmid containing the El region of human adenovirus 5 (WO 97/00326). PER cells are specifically useful in preventing the formation of RCA in combination with novel El adenovirus vectors (WO 97/00326), which do not possess sequence overlap with the El region present in PER cells. In one aspect of the invention, PER cells are provided with the helper virus function additionally required through the infection of an adenovirus without El that does not contain sequence overlap with El sequences already present in PER cells, which leads to the formation of RCA. In another aspect of the invention, PER cells are provided with the auxiliary virus function additionally required through transfection with plasmid DNA containing the genes encoding the helper virus function, plasmids which do not contain sequence overlap with The sequences of El already present in PER cells, which leads to the formation of RCA. An example of such cell PER, PER6 has been deposited under accession number 96022940 ECACC at the Center for Applied Microbiology Research (CAMR). The most commonly used cell lines for the production of rAAC are HeLa and 293. Although these cell lines are widely used there are several inherent disadvantages to them. HeLa cells are derived from human cancer and thus contain one or more oncogenes in their DNA. It is conceivable that some of the chromosomal DNA is co-packed with the viral vectors i ^^ | . . . . . . -_ " . . . . ......... . . . . . . tr? t? ra? 1t_r produced in those cells and thus can end up in the target cells in the patient. Compared to HeLa cells, 293 cells have the advantage that they are not derived from human cancer. However, they are stably transfected with some adenovirus sequences and as a result they express El genes (Graham et al, 1977a). This expression of the El gene is sufficient for the production of recombinant AAV (Herzog et al, 1997, Snyder et al, 1997bM Shou et al, 1998). However, cell line 293 has a disadvantage. Not only the El region is stably integrated into the DNA of the cells. On the left side of the adenovirus genome it is known that the cell line contains at least 1-4344 sequences of adenovirus 5 containing the left ITR, the packaging signal, the El gene and the gene encoding protein IX (Louis et al. al, 1997). The presence of more than just El sequences leaves a significant region of overlap on both sides with the most commonly used adenovirus vectors or deletion mutants such as dl312 (Snyder et al, 1997b). The region of overlap is sufficient for homologous recombination between the most commonly used adenovirus vectors and the adenovirus 5 sequences in 293. Such an event of homologous recombination can lead to the undesirable generation of the adenovirus that competes for reproduction (RCA) (Hehir et al, 1996). Especially for large scale preparations the presence of RCA in adenovirus vector patterns without El is a problem (Imler et al, 1996; Lochmuller et al, 1994). The invention described in (Ferrari et al, 1996) and WO 96/40240 comprises the transfection of 293 cells with a DNA fragment of 35,000 bp isolated from DNA digested with Xbal of adenovirus dl309 to provide the auxiliary functions of adenovirus for production of recombinant AAV. This technique is not ideal since this fragment of Xbal has considerable overlap with the adenovirus sequences in 293, which allows the inadvertent generation of the adenovirus that competes for reproduction. Another disadvantage is that dl309 has a DNA insert in the E3 region. The refinement of the technique has led to the generation of adenovirus helper plasmids with deletions of adenovirus genes, while retaining the role of helper virus for the production of recombinant AAV (WO 97/17458, Ferrari et al, 1997; Li; et al, 1997; Xiao et al, 1998a). The use of those helper plasmids without adenovirus helper gene to prevent RCA is generally restricted to 293 cells. As indicated above, this cell line has several disadvantages, an additional disadvantage of cell line 293 is that it only expresses the El region. and thus the auxiliary function additionally required for the efficient and large-scale production of recombinant AAV, which needs to be provided separately. In addition, the culture of 293 cells is considered problematic. In another aspect of the invention, a packaging cell is provided which expresses the El and E2a regions of adenovirus, for example derived from the ts25 adenovirus. In this preferred embodiment of the invention, the functional expression of E2a can be synchronized to utilize the performance of recombinant AAV. The additionally required auxiliary functions are provided in the form of an adenovirus without El, E2a or in the form of plasmid DNA containing the genes coding for the helper virus function, while the helper adenovirus vector or the plasmid DNA does not contain superposition of sequence with the functions of the helper virus already present in the packaging cells of the present invention, which leads to the formation of RCA. In this preferred embodiment, additional E2a auxiliary function can be provided to the packaging cell provided that the method does not introduce sequence overlap with the El region already present in the packaging cells., which leads to the formation of RCA. In a preferred embodiment of the invention, expression of adenovirus late genes is represented essentially by intervening with the transcription of the late genes or by removing one or more of the genes encoding the DNA encoding the additional auxiliary function required. In another aspect of the invention, the cells of the invention are grown in large numbers for the production, harvest and purification of recombinant AAV. For the production of recombinant AAV the cells are supplied with the DNA of the recombinant AAV, the DNA containing the AAV rep and cap genes and the DNA containing the auxiliary virus functions. In a preferred embodiment of the invention The AAV rep and cap genes are physically linked to the plasmid DNA which provides the auxiliary function additionally required, so that they are present on one and the same molecule. The cells can be provided with the DNA necessary for the production of recombinant AAV just before 15 of the start of the production of recombinant AAV, in which case, for each production the cells need to be supplied with the DNA through a process. The process can be any suitable method for the transfection or infection of DNA in large numbers of cells. In a The particularly preferred embodiment of the invention the DNA required for the production of recombinant AAV is transfected into PER cells by means of poly (2- (dimethylamino) ethyl-10-4-aminobutyl) phosphazene or other poly (organo) phosphazenes. Alternatively, the parts of the 25 DNAs required for the production of recombinant AAV can be stably integrated into the chromosomal DNA of the PER cell. In another aspect of the invention the cells of the invention, the recombinant AAV is produced with the packaging cell of the invention growing in suspension cultures using completely defined serum free medium. In one embodiment of the invention, a method for generating a packaging cell containing all the auxiliary functions necessary for an AAV reproduction cycle is provided, so that the auxiliary functions do not contain overlays that lead to the formation of the competing helper virus. for the reproduction. In a preferred embodiment of the invention, the packaging cell 15 is stably transformed with the El region of adenovirus, which region does not contain superposition with the auxiliary functions additionally required. In a particularly preferred embodiment of the invention, the packaging cell is stably transformed with the El region and the E2a gene. In this particular embodiment of the invention, the E2a function can be activated or deactivated after a signal. In a preferred embodiment of this invention the E2a gene is derived from the mutant adenovirus H5tsl25, whereby such a signal is a change in temperature. In another particularly preferred embodiment of the invention ^! _ ^ _ l _ = _ SftfeS ^ íf ^ _ÍllL _ - __ 1_ -il ^ _ ** £ * _. . » »,. _A »< The packaging cell is stably transformed with the El region of adenovirus 5, the E2a gene and the adenovirus 5 region (Martinez et al, 1989) or the E4orf6 gene of adenovirus 5, or both. In this particularly preferred embodiment, the transcriptional activity of the VA region of adenovirus 5 and / or the E4orf6 gene of adenovirus 5 is regulated. This means that the transcriptional activity can be activated or deactivated after a signal. As used herein the term "additionally required auxiliary function" also refers to helper virus functions that allow the efficient (large-scale) production of the recombinant AAV, for which the coding genes are not stably integrated into the DNA of the recombinant AAV. the cell that produces recombinant AAV or for which additional expression is desired. Such additionally required auxiliary functions can be provided through which viral or non-viral method capable of transferring external genetic material to mammalian cells such as, but not limited to: genetic transfer mediated by poly (organ) phosphazenes, polyethylene imine, phosphate precipitation of calcium, electroporation, recombinant, lipid or liposome. In one embodiment of the invention a packaging cell is provided which requires only an additional AAV packaging function and an AAV vector ^ Jjg ^ jl ^ _ * _ j! _i_i recombinant for the production of recombinant AAV. The packaging cell comprises and provides the required adenovirus helper function of the stably integrated adenoviral DNA. In one aspect of the invention the auxiliary function is provided by a stably integrated region 1. In another aspect of the invention, the helper function is provided by a stably integrated region and a stably integrated E2a gene. In this particular embodiment of the invention, the E2a function can be activated or deactivated after a signal. In a preferred embodiment of this invention the E2a gene is derived from the mutant adenovirus H5tsl25, whereby the signal is a change in temperature. In another particularly preferred embodiment of the invention, the packaging cell is stably transformed with the El region of adenovirus 5, the E2a gene and the VA region of adenovirus 5 (Martínez et al, 1989) or the E4orf6 gene of adenovirus 5, or both In this particularly preferred embodiment the transcriptional activity of the VA region of the adenovirus 5 and / or the E4orf6 gene of the adenovirus 5 is regulated, which means that the transcriptional activity can be activated or deactivated after a signal. The invention provides a cell culture comprising a cell according to the invention. Large-scale production of recombinant vectors for human gene therapy requires an easy and up-scalable culture method for the producer cell line, preferably a suspension culture or other such large-scale culture as a bioreactor culture, in medium 5 devoid of any human or animal constituents, ie in serum-free medium. Several "systems for growing mammalian cells in large numbers have been devised." These include, but are not limited to, rotary bottle culture, cellular juices and bioreactors. 10 of those systems have advantages and disadvantages. Bioreactors in which cells grow in suspension are the easiest to standardize and scale to ever larger volumes. However, a disadvantage is that the cells in suspension are not easily transfected. HE 15 developed many different cell culture media to support the optimal growth of a large variety of different cells. Most of these media are based on variations of Dulbecco's modified Eagle's medium (DMEM) and are supplemented with bovine serum. We have adapted 20 cells according to the invention to suspension cultures using a defined serum-free medium. The cultures free of serum have the advantage that they are completely defined since they do not depend on a natural source of serum that can vary in quality and presence of agents 25 adventitious. These serum-free media contain additives AIÉÍltAÉ that replace essential components for the growth of cells in serum. The invention also provides a method for producing recombinant adeno-associated viruses, comprising 5 using a cell or cell culture according to the invention and which provides for the use of those adeno-associated virus vectors in gene therapy. The invention is described on the basis of AAV-2 but it is clear that also other AAV serotypes (such 10 as 1 and 3 to 5) or even serotypes to be discovered can be adapted for the same purposes. Also the dependent viruses common in other species can be used for the same purposes, for example the canine adeno-associated adenovirus is capable of infecting cells 15 human. In addition, AAV reproduces in many types of mammalian cells, as long as the specific adenovirus species is present, and viruses dependent on other species can be produced with the cells and methods of the present invention using the respective species of 20 specific adenoviruses. Non-limiting examples of non-primate viruses are adeno-associated viruses of birds, canines, cattle (Berns, 1996). For example, it is clear to those skilled in the art that also adenovirus 1 to 4, 6 to 51 or other adenoviruses 25 humans or animals can be manipulated for the same .. ,,, ^ U - ^ _ eá¿-atete.f .: purpose, provided that the function between the genetic products is comparable. Genetic products that provide a similar AAV helper function but that are derived from different viruses, such as, but not limited to HSV, CMV and pseudorabies virus, or are derived from other natural sources or are produced in synthetic form, can be used for the same purpose. The invention is further explained in the experimental part of the description and the drawings without limiting the invention.

Brief description of the drawings Figure 1. Describe the structure and organization of the genome of the tAAV. Figure 2. PER.C6 cells were seeded at a density of 1 x 106 cells per 25 cm2 of cell culture flask and cultured at 32-, 37- or 39 ° C. At the indicated time points, the cells were counted in a Burker cell counter. PER.C6 cells grow both at 32-, 37- and 39 ° C. Figure 3. Western blot with 35mg of whole cell extract from cell lines generated from PER.C6 transfected with pcDNA3 (top panel, lane 1), pcDNA3wtE2A (top panel, lane 2), pcDNA3tsE2A (top panel, lanes 4-14; middle panel, lanes 1-13 and lower panel, lanes 1-12) or PER.C6 cells transiently transfected with pcDNA3tsE2A (upper panel, lane 3). The spot was probed with an antibody specific for the E2A gene product (B6 aDBP) and visualized using the ECL detection system. All PER.C6tsE2A cell lines express the temperature-sensitive DBP protein encoded by tsE2A. Figure 4. The cell line PER.C6tsE2A. c5-9 expressing tsE2A was cultured in suspension in serum free Ex-cellMR. The points in time indicated, the cells were counted in a Burker cell counter. The results of 8 independent cultures are indicated. PER.C6tsE2A grow well in suspension in serum-free Ex-cellMR medium.

Experimental part Materials and methods DNA constructs The packaging plasmid pIM45 (7.3 kb) contains the AAV-2 rep and cap genes (McCarty et al, 1991) and was a kind donation from Dr. S. Zolotukhin. The pACV-ßgal (8.3 kb) is a plasmid containing a CMV-LacZ expression cassette between AAV-ITRs and was a kind donation from Dr. J.A. Kleinschmidt. Plasmid pIG.ElA.ElB contains the Ela and El5 genes (nucleotides 459 to 3510 of Ad5) under the transcriptional control of the human PGK promoter and is described in WO / 97/00326. Plasmid pE2a is another name of plasmid pcDNA3wtE2A described below. Plasmid pE4orf6 was generated by inserting a 929 bp fragment encoding the E4orf6 protein of 10 Ad5 at the BamHI site of pCMV / neo (Hinds et al, 1990). pBr / Ad.Bam-rITR (ECACC deposit p97082122) To facilitate the cloning of the cut end of 15 ITR sequences, adenovirus type 5 DNA was treated (Ad5) natural human with Klenow enzyme in the presence of an excess of dNTPs. After the inactivation of the enzyme Klenow and purification by extraction with phenol / chloroform followed by ethanol precipitation, the DNA was digested with 20 BamHI. This DNA preparation was used without further purification in a ligation reaction with pBR322 derived from the vector DNA prepared as follows: pBR322 DNA was digested with EcoRV and BamHI, dephosphorylated by treatment with TSAP enzyme (Life Technologies) and purified 25 on LMP agarose gel (SeaPlaque GTG). After the Transformation into competent E. coli DH5a (Life Techn.) and analysis of ampicillin-resistant hills, a clone was selected that showed a digestion pattern as expected by an insert extending from the BamHI site on the Ad5 towards the ITR on the right. Sequence analysis of the cloning limit in the right ITR revealed that the G 3 'residue of the ITR was absent, the rest of the ITR was found to be correct. pBr / Ad.Cla-Bam (ECACC deposit P97082117) Native type 5 adenovirus DNA was digested with Clal and BamHI, and the 20.6 kb fragment was isolated from the gel by electroelution. PBR322 was digested with the same enzymes and purified from agarose gel by GeneClean. Both fragments were ligated and transformed into competent DH5a. The resulting clone pBr / Ad. Cla-Bam was analyzed by digestion with restriction enzyme and showed to contain an insert with adenovirus sequences from 919 to 21566 bp. pBr / Ad.AflII-Bam (ECACC deposit P97082114) The clone pBR / Ad. Cla-Bam was linearized with EcoRI (in pBR322) and partially digested with AflII. After thermal inactivation of AflII for 20 'at 65 ° C the ends of the fragment were filled with Klenow enzyme. j ^^ - ^ ^ The DNA was then ligated to a double-strand binder oligo that contained a Pací site (5'- AATTGTCTTAATTAACCGCTTAA-3 '). This binder was produced by annealing the following two oligonucleotides: 5'- 5 AATTGTCTTAATTAACCGC-3 'and 5' -AATTGCGGTTAATTAAGAC-3 ', followed by cutting with Klenow on top. After precipitation of the DNA bound to the change buffer, the ligations were digested with an excess of PacI enzyme to remove concatemers from the oligo. The partial fragment of 22016 10 bp containing Ad5 sequence from 3534 to 21566 bp and vector sequences, was isolated on LMP agarose (SeaPlaque GTG), religated and transformed into competent DH5a. A clone that was found contained the Pací site and that had retained the large adenofragment was selected and sequenced 15 at the 5 'end to verify the correct insertion of the Pací linker at the AflII site (lost). pBr / Ad.Bam-r! TRpac # 2 (ECACC deposit P97082120) and pBr / Ad.Bam-rITR # 8 (ECACC deposit P97082121) 20 To allow insertion of the Paci site near the Ad5 ITR in the clone pBr / Ad. Bam-rITR approximately 190 nucleotides were removed between the Clal site in the pBr322 backbone and the start of the ITR sequences. This was done as follows: pBr / Ad was digested. Bam-rITR with - ^ ammUmmem * ** - Clal and treated with nuclease Bal31 during different periods of time (2 ', 5', 10 'and 15'). The degree of nucleotide removal was followed by separate reactions on the DNA of pBR322 (also digested at the Clal site), using different buffers and conditions. The Bal31 enzyme was inactivated by incubation at 37 ° C for 10 ', the DNA was precipitated and resuspended in a smaller volume of TE buffer. To secure the cut ends, the DNAs were further treated with T4 DNA polymerase in the presence of an excess of dNTPs. After digestion of pBr322 (control) DNA with Sali, satisfactory degradation (~ 150 bp) was observed in the treated sample for 10 'or 15'. The pBr / Ad.Bam-rITR samples treated for 10 'or 15' were then bound to the cut Paci binders described above (see pBr / Ad. Aflll-Bam). The ligations were purified by precipitation, digested with excess Paci and separated from the binders on an LMP agarose gel. After religation, the DNAs were transformed into competent DH5a and the colonies were analyzed. Ten clones were selected that showed a deletion of approximately the desired length and those were further analyzed by sequencing of T tracking (T7 sequencing kit, Pharmacia Biotech). Two clones were found with the Pací linker inserted just downstream of the rITR. After the Bt «? In the first digestion with Pací, clone # 2 had 28 bp and clone # 8 had 27 bp joined to the ITR. pBr / Ad.aflII-rITR (ECACC deposit P97082116) 5 Cosmetic vector pWE15 (Clontech) was used to clone larger Ad5 inserts. First, a linker containing a single PacI site was inserted into the EcoRI sites of pWE15 creating pEW15.Pac. To this end, the double-stranded Pacigo oligo was used as described for 10 pBr / Ad. Af111-Bam but not with its EcoRI projection ends. The following fragments were then isolated by electroelution from agarose gel: pWE15.Pac digested with Pací, pBr / Ad. Af Hl-Bam digested with Pací and BamHI and pBr / Ad. Bam-rITR # 2 digested with BamHI and Pací. Those fragments 15 were ligated together and packaged using phage packaging extracts? (Stratagene) according to the manufacturer's protocol. After infection in host bacteria, the colonies were grown on plates and analyzed for the presence of the complete 2Q insert. The pWE / Ad. AfHl-rlTR contains all type 5 adenovirus sequences of 3534 bp (AflII site) up to and including the right ITR (missing G plus 3 'residue).

--- -M "u'j < iw *! - -. .-- --- ^. -. ~~ -. - ^ -. .. - -, _ *« ^ * - rf- a p E / Ad. 5 'The pWE / Ad.? 5' construct is an example of a reproduction molecule according to the invention containing two adenoviral ITRs and all the adenoviral sequences between 3510 bp and 35938 bp, the complete adenoviral genome except for the El region and the packaging signal.PWE / Ad.? 5 'has been produced in a three fragment cosmid vector background First the ITR5' of Ad5 was amplified using the following primers : ITR-EPH: 5 '-CGG-AAT-TCT-TAA-TTA-AGT-TAA-CAT-CAT-CAA-TAA-TAT-ACC-3' and ITR-pIX: 5 '-ACG-GCG-CGC- CTT-AAG-CCA-CGC-CCA-CAC-ATT-TCA-GTA-CGT-ACT-AGT-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3. 'The resulting PCR fragment was cloned into vector pNEB193 (New Enland Biolabs) digested with the same enzymes The resulting construct was named pNEB / ITR-pIX, and sequencing confirmed the correct amplification of the Ad5 sequences in the Left ITR (Ad5 sequences 1 to 103) linked to the pIX promoter (Ad5 sequences 3511 to 3538) except for a single disparity with the expected sequence according to GenBank (No. of access: M73260 / M29978), that is, that an extra G residue was found just upstream of the AflII site. This fragment of ITR-pIX was then isolated with EcoRI-AflII and ligated to a fragment of the EcoRI-AflII vector containing 3539-21567 ^^ Se ^^ leséi ^ sequences of Ad5. The last fragment was obtained by digestion of pBr / Ad. Cla-Bam (supra) with EcoRI and partially with AflII. The resulting clone was named pAd / LITR (? 5 ') -BamHI. The final construct pWE / Ad.? 5 'was then produced by ligating the cosmid vector pWE15.Pac (supra) digested with PacI to pAd / LITR (? 5') -BamHI digested with PacI / BamHI and pBr / Ad. Bam-rITR. pac # 2 (supra) digested with PacI / BamHI. pWE / Ad, AflII-rITR? E2A. This success is essentially the same as the pWE / Ad.AflII-rITR (ECACC deposit P97082116) plus a deletion of the E2A coding regions. The deletion of the sequences coding for E2A from pWE / Ad.AflII-rITR (ECAAC deposit P97082116) was carried out as follows. The adenoviral sequences flanking the region coding for E2A on the left and right side were amplified from the pBR / Ad plasmid. Salt . rITR (ECACC deposit P97082119) in a PCR reaction with the Expand PCR system (Boehringer) according to the manufacturers protocol. The following primers were used: Right flanking sequences (corresponding to nucleotides 24033 to 25180 of Ad5):? E2. SnaBI: 5 '-GGC. GT. CGT .AGC. CCT. GTC GAA.AG-3 ' I. »ít -,,. , ." - TO.

? E2A. DBP-Start: 5 '-CCA.ATG. CAT.TCG.AAG. TAC TTC CTT. CTC CTA. TAG GC-3 'The amplified DNA fragment was digested with SnaBI and Nsil (Nsil was generated from the primer? E2A. DBP-start, underlined) The left flanking sequences (corresponding to nucleotides 21557 to 22442 of Ad5):? E2A. DBP-stop: 5 '-CCA.ATG. CAT .ACG. GCG CAG .ACG. G-3 ' ? E2A.? AmHI: 5 '-GAG. GTG GAT CCC.ATG. GAC GAG-3 'Amplified DNA was digested with BamHI and Nsil (Nsil was generated in the primer? E2A. DBP-stop, underlined). Subsequently, the digested DNA fragments were ligated into pBr / Ad. Sal-rITR digested with SnaBI / BamHI to give rise to pBE.Ad. Sal-rITR? E2A. The sequencing confirmed the exact replacement of the region coding for DBP with a unique Nsil site in the plasmid pBR.Ad.Sal-rITR? E2A. Next, the cosmid pWE / Ad.AfIII-rITR? E2A was generated. The plasmid pBR.Ad. Sal-rITR? E2A was digested with BamII and Spel. The 3.9 kb fragment in which the region coding for E2A was replaced by the unique Nsil site was isolated. The cosmid pWE / Ad.Af111-rITR was digested by BamHI and Spel. The 35 Kb DNA fragment, of which the ^ á.

BamHI / Spel fragment containing the sequence coding for E2A was removed, was isolated. The fragments were ligated and packaged using phage packaging extracts? according to the protocol of the manufacturers (Stratagene), producing the cosmid pWE / Ad.AflII-rITR? E2A. pVA. The pVA (3.7 Kb) is a pUC119 plasmid that contains the VAI and VAII region of adenovirus 5 (nucleotides 10555 to 11075). The VA genes of adenovirus 5 were cloned after PCR on the DNA isolated from the native adenovirus 5 using the primers 5'-ACGCGTCGACCTCTGGCCGGTCAGGCGCGCGCAA-3 'and 5'-ACGCGGCTCCCGCATCTGCCGCAGCACCGGATGC-3'. The PCR was carried out using the PCR eqμipo expand long template ™ (Boehringer) according to the manufacturer's specifications. The resulting fragment was digested with Sali, and BamHI, present in the primers, and ligated in pUC119 digested with Salí and BamHI.

Cell Culture Cells 293 (Graham et al, 1977b) and HeLa cells (Cancer Res. 12: 264, 1952) were cultured in medium -ÁÁ .I Dulbecco modified Eagle (DMEM, Life technologies Breda, The Netherlands) containing 10% heat inactivated fetal bovine serum at 37 ° C and 10% C02. The adherent cultures of PER.C6 cells were grown in DMEM supplemented with 10% fetal bovine serum and MgCl2 (10 mM) at 37 ° C and 10% C02. Suspension cultures of PER.C6 cells were grown in Ex-Cell ™ 525 (JRH Biosciences, Denver, Pennsylvania) supplemented with 1 x L-Glutamine (GIBCO BRL), hereinafter referred to as Ex-Cell ™ at 37 ° C and 10 ° C. % of C02 in stationary cultures in 6-well disks (Greiner, Alphen aan de Rijn, The Netherlands) or in Erlenmeyer tissue culture vessels (Corning) during continuous agitation at 100 RPM.

Transfection Transfection of monolayer cultures: Cells HeLA and 293 cells were transfected using the Calcium Phosphate transfection system (Life technologies, Almere) according to the manufacturer's specifications. The monolayers of PER.C6 cells were transfected using LipofectAMINEMR (Life technologies, Breda) according to the manufacturer's specifications. Transfection of cultures in suspension: 'The PER.C6 cells in the logarithmic growth phase were collected by centrifugation (3000 g, 5 minutes, rta). The cells were resuspended in transfection mixture (described below at a concentration of 2 x 106 and incubated for 3 hours at 37 ° C, 10% C02.) Unless otherwise indicated, transfections were performed under shaking conditions. (100 RPM) For transfections with DMRIE-CMR 5 (Life technologies, Breda) the transfection mixture was made in DMEM according to the manufacturer's specifications After the 3-hour incubation in the transfection mixture, the cells were collected by centrifugation (3000 g, 5 min, rta) and resuspended in medium 10 Ex-CellMR fresh to a final concentration of 106 cells per ml. Transfection with FuGENEMR (Boehringer Mannheim) was carried out with a transfection mixture made in the Ex-Cell ™ medium according to the manufacturer's specifications. After three hours of incubation with transfection mixture the 15 cells were diluted with Ex-Cell ™ medium to a final concentration of 106 cells per ml. The trinfection mixtures using poly (2- (dimethylamino) ethyl-10-4-aminobutyl) phosphazene (PPZ) were made as follows. A standard PPZ solution (2.4 mgrams / ml) was produced by dissolving the 20 solid compound in Hepes (5 mM, pH = 7.3). The PPZ formula is: Xi I - [P = N-] nP = N- where Xl f X2 are -N-CH2-CH2-N (CH3) 2 or -N-CH2- 25 CH2-CH2-CH2-NH2 .

X2 The transfection mixtures were made by adding the indicated amount of PPZ to 500 ul of Ex-Cell ™ medium. This solution was mixed with the same volume of Ex-Cell ™ containing the indicated amount of DNA. The mixture was incubated for 1 hour and subsequently used to resuspend a sediment of 2 x 10 6 cells of PER.C6. The cells were incubated with the transfection mixture for three hours and then diluted with Ex-Cell ™ medium to a final concentration of 10 6 cells per ml. The transfected cells were harvested 48 hours later and analyzed for β-galactosidase activity.

Assays activity of β-galactosidase The cells were stained for β-galactosidase activity with two different methods. For the histochemical analysis and the determination of the number of infectious units, the following procedure was used. Cells were washed twice with PBS (NPBI, Emmer-Compascuum) and fixed for 10 minutes in 0.2% glutaraldehyde (Sigma, Zwijndrecht, The Netherlands) in PBS. Cells were washed twice with PBS and stained with X-Gal solution > ^ -1 - J "» - • «• - - - - - - _ T tm 1- -",. ^., ^^ (MgCl2.6H20 2 mM, K2Fe (CN) 6 5 mM, 'K4Fe (CN) 6.3H20 5 mM and 40 mg / ml X-Gal (5-bromo-4-chloro-3-indolyl-b-galactopyranoside, Molecular Probes Europe, Leiden, The Netherlands) in 0.1 M phosphate buffer pH = 7.4 After the tinsion during 5 night at 37 ° C the blue cells were counted under an optical microscope (Olumpus CK2-TR) For the quantification of the activity of the β-galactosidase in suspension cultures of PER.C6. used the LacZ Quantification / Galactosidase FluoReporter ™ (Molecular Proes, Leiden, Holland) equipment according to the protocol provided by the manufacturer The β-galactosidase activity of each sample was evaluated by comparing the β-galactosidase activity in 10 samples. ul of the cell suspension at a serial dilution of a known concentration of purified β-15 galactosidase.

Titration of recombinant AAV HeLa cells were seeded at 4 x 104 cells per cm2. The medium was replaced the next day with fresh medium containing serial dilutions of rAAv and adenovirus tsl49 (20 pfu / cell). After 4 hours the medium was replaced by fresh medium and the cells were incubated for 24 hours at 37 ° C, 10% CO2 before tinsion with β-galactosidase. The title of the recombinant AAV standard was ^ ^ | j & ^ g calculated by counting the number of blue cells of the highest dilution that gave rise to the blue cells and multiplying this number by the dilution factor. 5 Production of recombinant AAV on adherent cells The cells were seeded so that they reached a confluence of 70% the following day. The cells were then transfected with pACV-ßgal and pIM45 (ratio 1: 4 weight / weight) and infected with the adenovirus helper virus 10 tsl49 (moi = 20), the adenovirus helper virus without the IG.Ad.MLP.Luc (Vincent et al., 1996) or transfected with the adenovirus helper plasmid DNA. In the latter case the total amount of plasmids of the adenovirus helper gene was 1.5 times (weight / weight) greater than the total amount of pACV-ßgal and pIM45. When more than one adenovirus helper plasmid was used, equal amounts (weight / weight) of the different adenovirus plasmids were used. The production of recombinant AAV on PER: C6tsE2A. c5-9 adherent was infected according to that described for cell line PER.C6 except for some modifications. The cell line was grown at 39 ° C, 10% Co2. Before transfection the cells were seeded at 39 ° C, 10% C02, so that they reached a confluence of 7%) the next day. The cells were subsequently cultured during a J ^ m ^ gj ^ fcjj ^^ _ ^ __- ^ __ ^ _ ^ _ ^ • _. . . I ^ aS? ^, J day at 32 ° C, 10% C02. The cells were then transfected at 37 ° C, 10% Co2 according to what was described for the PER.C6 cell line. The recombinant AAV was harvested 48 hours after transfection. The cells 5 were detached in their culture medium and subjected to three cycles of freezing and thawing. Cell debris was centrifuged (200 RPM, 10 minutes, ct). When adenovirus tsl49 or adenovirus vectors without El were used, the supernatants were inactivated with 10 heat at 56 ° C for 1 hour. When adenovirus DNA fragments were used to complement the production of AAV the supernatants were not thermally inactivated. All supernatants were filtered (0.45 uM, Millipore) before storage at -20 ° C. Example 1 Generation of producer cell lines for the production of recombinant adenoviral vectors without the initial region 1 and the initial region 2A 20 The generation of cell lines for the production of recombinant adenoviral vectors without the initial region 1 (El) is described here and the initial region 2A (E2A). Producer cell lines complement the deletion of El and E2A from the recombinant adenoviral vectors in the trans position by the constitutive expression of the El and E2A genes, respectively. The human embryonic kidney retinoblastic cell line transformed from the pre-established Ad5 Ad.C6 (WO 97/00326) and the human embryonic kidney cell line transformed with Ad5 293 (Graham et al, 1977b) were further equipped with E2A expression cassettes. . The E2A adneoviral gene codes for a 72 kDa DNA Binding Protein (DBP) which has a high affinity for single-stranded DNA. Due to this characteristic, the constitutive expression of DBP is toxic to cells. The tsl25E2A mutant codes for a DBP which has a Pro? Ser substitution of amino acid 413 (Vliet van der et al, 1975). Due to this mutation, the DBP encoded by tsl25E2A is fully active at the permissive temperature of 32 ° C but does not bind to the ssDNA at the permissive temperature of 39 ° C. This allows the generation of cell lines that constitutively express E2A, which are not functional and are not toxic at the non-permissive temperature of 39 ° C, but become functional after a temperature change at the permissive temperature of 32 ° C.

^? .... . . . A. Generation of plasmids expressing natural E2A or tsl25E2A sensitive to temperature. pcDNA3wtE2A: The region encoding the complete native 2A (E2A) region was encoded from the plasmid pBR / Ad.Bam-rITR (ECACC deposit P97082122) with the primers DBPpcrl and DBPpcr2 using the large-pattern PCR system ExpandMR according to standard protocol of the distributor (Boheringer Mannheim). The PCr was carried out in a Biometra Trio Thermoblock amplification program: 94 ° C for 2 minutes, 1 cycle; 94 ° C for 10 seconds + 51 ° C for 30 seconds + 68CC for 2 minutes, 1 cycle; 94 ° C for 10 seconds + 58 ° C for 30 seconds + 68 ° C for 2 minutes, 10 cycles; 94 ° C for 10 seconds + 58 ° C for 30 seconds + 68 ° C for 2 minutes with 20 seconds extension per cycle, 20 cycles; 68 ° C for 5 minutes, 1 cycle. The DBPpcrl: CGG GAT CCG CCA CCA TGG CCA GTC GGG AG AGG (5 'to 3') primer contains a unique BamHI restriction site (underlined) 5 'of the Kozak (italic) sequence and the start codon of the sequence that codes for E2A. The DBPpcr2 primer: CGG AAT TCT TAA AAA TCA AAG GGG TTC TGC CGC (5 'to 3') contains a unique EcoRI restriction site (underlined) 3 'and the stop codon of the sequence coding for E2A. Bold characters refer to the sequences derived from the region coding for E2A.

The PCR fragment was digested with Ba HI / EcoRI and cloned into pcDAN3 digested with BamHI / EcoRI (Invitrogen) to give pcDNA3vtE2A. pcDNA3tsE2A: The coding region of tsl25E2A 5 was amplified from the DNA isolated from the temperature sensitive adenovirus mutant H5tsl25 (Ensinger and Ginsberg, 1972; Vliet van der et al, 1975). The PCR amplification procedure was identical to that of the wtE2A amplification. The PCR fragment was digested with BamHI / EcoRI and cloned in 10 pcDNA3 digested with BamHI / EcoRi (Invitrogen), giving rise to pcDNA3tsE2A. The integrity of the coding sequence of wtE2A and tsE2A was confirmed by sequencing.

B. Growth characteristics of producer cells 15 for the production of recombinant adenovirus vectors grown at 32-37- and 39 ° C. PER.C6 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO BRL) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO BRL) and 10 mM MgCl2 20 in an atmosphere with 10% Co2 at 32 ° C, 37 ° C or 39 ° C. On day 0, a total of 1 x 106 PER.C6 cells were sealed per 25 cm2 of tissue culture flask (Nunc) and the cells were cultured at 32 ° C, 37 ° C or at 39 ° C. At day 1-8 the cells were counted. Figure 3 shows that the speed of growth and the final cell density of the PER.C6 culture at 39 ° C is comparable to that of 37 ° C. The growth rate and final crop density of PER: C6 at 32 ° C were slightly reduced compared to 37 ° C or 39 ° C. No significant cell death was observed at any of the incubation temperatures. In this way the PER.C6 work very well both at 32 ° C and at 39 ° C, the permissive and non-permissive temperature for tsl25E2A, respectively.

C. Transfection of PER. C6 and 293 with E2A expression vectors; transformation and generation of cell line colonies One day before transfection, 2 x 10 6 PER.C6 cells were seeded per 6 cm of tissue culture dish (Greiner) in DMEM, supplemented with 10% FBS and MgCl2 lOmM and incubated at 37 ° C in an atmosphere with 10% C02. The next day, the cells were transfected with 3-, 5- u 8μg of either plasmid DNA from pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A per disc, using the LipoFectAMINE PLUSMR reagent kit according to the distributor's standard protocol (GIBCO BRL), except that the cells were transfected at 39 ° C in an atmosphere with 10% of C02. After transfection, the cells were constantly maintained at 39 ° C, the temperature not permissive for tsl25E2A. Three days later, the cells were placed on DMEM, supplemented with 10% FBS, MgCl2 lOmM and 0.25mg / ml of G418 (GIBCO BRL) and the first G418-resistant colonies appeared 10 days after transfection. As shown in Table 1, there was a dramatic difference between the total number of colonies obtained after transfection of pCDNA3 (~ 200 colonies) or pcDNA3stE2A (-100 colonies) and pcDNA3wtE2A (only 4 colonies). These results indicate that the constitutively expressed E2A toxicity can be overcome by using a temperature sensitive E2A mutant (tsl25E2A) and culturing the cells at the non-permissive temperature of 39 ° C. From each transfection, a number of colonies were taken off the disc cells with a pipette. The detached cells were then placed in 24 well tissue culture dishes (Greiner) and further cultured at 39 ° C in an atmosphere with 10% C02 in DMEM, supplemented with 10% FBS, MgCl2 lOmM and 0.25 mg / ml of G418 As shown in Table 1, 100% of the colonies transfected with pcDNA3 (4/4) and 82% of the colonies transfected with pcDNA3tsE2A (37/45) were established to stable cell lines (the remaining 8 colonies transfected with pcDNA3tsE2A grew slowly and were discarded). In contrast, only one colony transfected with ,., ... ... .. ****** pcDNA3wtE2A could be established. The other three died directly after being removed. Next, the expression levels of E2A in the different cell lines were determined by Western electro-Western blotting. The cell lines were seeded on 6-well cell culture dishes and the subconfluent cultures were washed twice with PBS (NPBI) and used and detached in RIPA (1% NP-40, 5% sodium deoxycholate and 0.1% SDS in PBS, supplemented with phenethylsulfonylfluoride lmM and 0.1 mg / ml trypsin inhibitor). After 15 minutes of incubation on ice, the used ones were clarified by centrifugation. The protein concentrations were determined by the Bio-Rad protein assay, according to the distributor's standard procedures (BioRad). Equal amounts of fractionated whole cell extract were fractionated by SDS-PAGE on 10% gels. The proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the a6B monoclonal antibody B6 (Reich et al, 1983). The secondary antibody was a goat anti-mouse antibody conjugated with horseradish peroxidase (BioRad). Western electroblotting procedure and incubations with antibody were performed according to the protocol provided by Millipore. The antibody complexes were visualized with the CT detection system, according to the manufacturer's protocol (Amersham). Figure 3 shows that all cell lines derived from transfection with pcDNA3tsE2A express the 72 kDa E2A protein (upper panel, lanes 4-14, middle panel, lanes 1-13, lower panel 5, lanes 1-12). In contrast, the only cell line derived from transfection with pcDNA3wtE2A did not express the E2A protein (lane 2). No E2A protein was detected in the extract of a cell line derived from the transfection with pcDNA3 (lane 1), which serves as a negative control. The extract of PER.C6 cells transiently transfected with pcDNA3tsl25 (lane 3) served as a positive control for the Western electro-Western blot procedure. These data confirm that the constitutive expression of wtE2A is toxic to cells 5 and that this toxicity can be circumscribed using the tsl25 mutant of E2A. In contrast to PER.C6 cells, the culture of 293 cells at 39 ° C is problematic. Therefore, transfection of 293 cells with pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A was carried out at 37 ° C in an atmosphere with 10% C02, a semipermissive temperature for the DBP encoded by tsl25E2A. One day before transfection, 293 cells were seeded in, supplemented with 10% FBS and MgCl2 lOmM, at a density of 3.6 x 10 5 cells per cm tissue culture dish (Greiner). Five hours before the g ^ | g ^^ g¡ ^ | s transfection, the cells received fresh medium. The cells were transfected with 7.2 μg of plasmid DNA from pcDNA3wtE2A or pcDNA3tsE2A, using the calcium phosphate transfection system according to the standard protocol of the distributor (GIBCO BRL). Two days after transfection, the cells were placed on selection medium, ie DMEM supplemented with 10% FBS, MgCl2 lOmM and O.lmg / ml of G418. The first colonies appeared 12 days after transfection. As shown in Table 10 2, the total number of colonies obtained after transfection of pcDNA3 (-100 colonies) or pcDNA3tsE2A (-25 colonies) was significantly greater than that obtained after transfection of pcDNA3wtE2A (only two colonies) . These results again show that E2A 15 constitutively expressed is toxic to cells, and that this toxicity can be circumscribed using tsl25E2A. In addition, it shows that this is not specific for PER.C6 cells, but that it is applied to eukaryotic cells in general (eg 293 cells). 20 D. Complementing the deletion of E2A in Ad5. dl802 by PER cells. C6 constitutively expressing tsl25E2A. The adenovirus? D5. dl802 is a vector derived from Ad5 for the main part of the region that codes for the• - "a» -t »Wfci-? Mili i * • • *" -'- '* E2A and that does not produce functional DBP (Rice and Klessig, 1985). Ad5.dl802 was used to test the transcomplementation activity of E2A of PER.C6 cells constitutively expressing tsl25E2A. The original PER.C6 cells or 3-9 clones of PER.C6tsE2A were cultured in DMEM, supplemented with 10% FBS and MgCl2 lOmM at 39 ° C and 10% C02 in bottles at 25 cm2 in cells pseudoinfected or infected with Ad5 .dl802 to moi of 5. Subsequently the infected cells were cultured at 32 ° C and the cells were separated by their appearance of a cytopathic effect (CPE) as determined by changes in cell morphology and cell detachment from the flask. Table 3 shows that the complete CPE appeared in the 3-9 clones of PER.C6tsE2A infected with Ad5.dl802 within 2 days. No CPE appeared in PER.C6 cells infected with AD5.dl802 or pseudoinfected cells. These data show that PER.C6 cells constitutively express the complement of tsl25E2A in trans for the suppression of E2A in vector Ad5.dl802 at the permissive temperature of 32 ° C.

E. Culture in serum-free suspension of PER cell lines. C6tsE2A. The large-scale production of recombinant adenoviral vectors for human gene therapy requires an easy and scalable culture method. »...,, ... », -. . ....... __ ^ ___, .s? ass. ascending for the producer cell line, preferably in suspension culture, in medium without any human or animal constituent. Up to that point, several clones of PER.C6tsE2A were taken to a suspension culture. As an example, the cell line PER.C6tsE2A (designated c5-9) was cultured at 39 ° C and 10% C02 in a 175 cm2 tissue culture flask (Nunc) in DMEM, supplemented with 10% FBS and MgCl2 lOmM. In the subconfluence (70-80% confluent), the cells were washed with PBS (NPBI) and the medium was replaced with 25 ml of serum-free suspension medium Ex-CellMR 525 (JRH) supplemented with 1 X of L-Glutamine (GIBCO BRL), hereinafter designated as Ex-CellMR. Two days later, the cells were detached from the flask by oscillation and the cells were centrifuged at 100 rpm for 5 minutes. The cell mass was resuspended in 5ml of Ex-Cell ™ and 0.5ml of cell suspension was transferred to a 80 cm2 tissue culture flask (Nunc), along with 12 ml of fresh Ex-Cell ™. 2 days later, the cells were harvested (all cells are in suspension) and counted in a Burker cell counter. The cells were then seeded in a 125ml tissue culture Erlenmeyer (Corning) at a seeded density of 3 x 10 5 cells per ml in a total volume of 20 ml of Ex-Cell ™. Cells were further cultured at 125rpm on an orbital shaker (GFL) at 39 ° C t ^ .......... ..., ......._ ^. . .. . _______-, -m? Mn in an atmosphere with 10% C02. The cells were counted on day 1-6 in a Burker cell counter. In Figure 4, the average growth curve of 8 crops is shown. PER.C6tsE2A works well in free suspension culture 5 of serum. The maximum cell density of approximately 2 x 10 6 cells per ml was reached within 5 days of culture.

Example 2 PER cells as producing cells for recombinant AAV. 10 PER cells were derived from human retinal cells. The retina is known for its ability to sustain the reproduction of AAV. Therefore, we verify if the PER cells are permissive for the production of recombinant AAV. PER.C6 cells were transfected using LipofectAMINAMR with the packaging plasmid pIM45, the rAAV vector pACV-ßgal (ratio 10: 1 wt / wt) and infected with tsl49 adenovirus. The recombinant AAV was isolated two days later and titrated on HeLa cells infected with adenovirus, the pACV-ßgal produced on the 20 PER.C6 cells had a titer of 2 x 107 infectious units (Ul) per ml -Q 20 Ul per cell. The virus yield per cell obtained with this system is comparable or better than that reported for the 293 cell lines with the packaging plasmid pIM45 (Vincent et al, 1997). In parallel J ^^ | f | ¡^ ^ ¿8fe - :. »We analyzed if the pattern and level of the expressed proteins was sufficient for the production of rAAV. To evaluate this question, the PER.C6 cells were transfected with the packaging plasmid pIM45, the 5 rAAV vector pACV-ßgal (ratio 10: 1 wt / wt) and infected with an adenovirus vector without IG. Ad.MLP. Luc (Vincent et al, 1996). The performance of rAAV using the adenovirus vector without the ID. Ad.MLP. Luc was 2 x 107 IU / ml and thus the same as with the tsl49 adenovirus. 10 This result indicates that both the pattern and the level of expression of El in PER.C6 allows the efficient production of rAAV.

Viral vector free production of recombinant AAV on PER.C6. The production of recombinant AAV using adenovirus DNA as an auxiliary in place of the adenovirus has been reported (Ferrari et al, 1997, Ferrari et al, 1996, Li et al, 1997, Xiao et al, 1998a)). This work was done exclusively in the 293 cell line. We wish To determine if also the PER.C6 cell line could be used to produce recombinant AAV. Up to this point we tested two constructs pWE / Ad.D5 'and pWE / Ad.Af111-rITR which contained all the adenovirus genes except the El region. The pWE / Ad construct. f111-rITR, as mentioned it contains all the adenovirus except El, however, the promoter of the protein IX gene contains a deletion. PW / Ad.D5 'on the other hand contains the left ITR and the entire length of the protein IX promoter. The recombinant AAV 5 could be produced with both adenoviral DNA fragments. The yields of recombinant AAV were different compared both constructs. The performance of the recombinant AAV using pWE / Ad. Af111-rITR was significantly higher than using pWE / Ad.D5 ' 10 (Table 4). One reason for the difference in performance could be the difference in the expression of the relevant proteins of the two different plasmids. Another reason could be that the expression of protein IX of adenovirus 5 adversely affects the production of recombinant AAV. 15 Minimum requirements for the production of recombinant AAV in PER.C6. To determine the minimum requirement for the production of recombinant AAV in the PER.C6 cell line We obtained plasmid clones for the adenovirus genes that are known to affect AAV production (ie, E2a, E4orf6 and VA). Together with the El region already present in PER.C6, all adenovirus genes that are known to affect AAV production were thus tested for their effect on 25 the production of recombinant AAV on PER.C6. By contrast with HeLa cells, PER.C6 cells produce a low but detectable amount of recombinant AAV when transfected with pIM45 and pACV-ßgal. The efficient production of the recombinant AAV required by transfection of genes coding for the additional auxiliary. The highest amount of recombinant AAV was obtained from the transfection of the three expression cassettes by pE2a and pVA (Table 4). Transfection of pE2a alone or together with pE4orf6 resulted in + 10% yield of the recombinant AAV compared to the other three genes. The transfection of only E4 or only VA did not produce a significant amount of AAV (Table 4). The production on PER. C6tsE2A. c5-9 without addition of extra adenovirus helper genes resulted in a titer of 1.3 x 103. With the addition of pE4orf6 and pVA alone or in combination with pE2A the yield of recombinant AAV was increased. The highest values were obtained using the pWE / Ad.Af111-rITR construct.

Large scale production of recombinant AAV Several systems have been devised to grow mammalian cells in large numbers. These include but are not limited to rotary bottle cultivation, cubes and cellular biorrectives. Each of these systems has advantages and disadvantages. The bioreactors in which . ^^ ¿¿.

They make growing cells in suspension are the easiest to standardize and scale to ever larger volumes. However, a disadvantage is that the cells in suspension are not easily transfected. Many different cell culture media were developed to support the optimal growth of a large variety of different cells. Most of these media are based on variations of Dulbecco's modified Eagle's medium (DMEM) and are supplemented with bovine serum. We have adapted PER.C6 cells to suspension cultures using a defined serum-free medium. The serum-free cultures have the advantage that they are completely defined since they do not depend on a natural source of serum that can vary in quality and presence of adventitious agents. These serum-free media comprise additives that replace essential components of cell growth in serum. We observed that many transfection reagents, specifically liposomes, function better when the cells were cultured in DMEM than when the cells were cultured in serum free Ex-Cell ™ medium (not shown). The transfection of PER.C6 cells could, however, be achieved using a non-liposomal reagent FuGENEMR or avoiding contact of the liposome: DNA complex (DMRIE-C) with the Ex-Cell ™ medium (Table 5). The alternative transfection agents are (organ) phosphazenes. Not much is known about the ability to transfect cells with these agents. To study the use of such agents for transfection of suspended PER cells, we transfected with increasing amounts of the compound poly (2- (dimethylamino) ethyl-10-4-aminobutyl) phosphazene (PPZ) with a constant amount of DNA. The cells were exposed to the transfection mixture for 3 hours before the removal of the medium. Transfection of the cells in suspension was measured by suspension with X-Gal and fluorometric analysis depending on the amount of PPZ added (Table 6 and 7) reaching 5% of positive X-Gal cells with 320 ug of PPZ (Table 6) and 160 ug of PPZ (Table 7). Larger amounts of PPZ in the transfection mixture were associated with an exhaustive loss of cells. Next, we evaluated the potential of PER cells for the large-scale production of recombinant AAV. Potential large-scale production was evaluated first on adherent PER.C6 cells. Next, 10 170 cm2 discs (Greiner) with 2 x 107 PER.C6 were seeded by diso in DMEM + 10% of CFS. The cells were transfected with pACV-ßgal, pIM45 and pWE / Ad. AfIII-rlTR (2: 8: 30 ugramo respectively). The viruses were harvested 48 hours later according to the following protocol. The medium was removed from the cells and the cells were collected by detaching them in 4 ml / disc in DMEM .JJ--. cool. The cell suspension was frozen and thawed twice and then incubated with Dnasel (100 ugram / ml) at 37 ° C, 30 minutes. The suspension was subjected to two additional freeze and thaw cycles after which the cell debris was removed by centrifugation (3000 RPM, 10 minutes). The supernatant was incubated with 13.3 ml of saturated (NH4) 2 SO4 (4 ° C, 10 minutes). The precipitate was removed by centrifugation at 10,000 RPM in a SW27.1 rotor (4 ° C, 15 minutes). The supernatant was incubated with an additional 26.6 ml (NH4) 2SO4 saturated and incubated for 20 minutes at 4 ° C. The virus was pelleted by centrifugation at 12.00 RPM in a SW27.1 rotor (4 ° C, 30 minutes). The pellet was suspended in 5 ml of PBS (NPBI) and equally divided into two Quick-Seal Ultra-Clear tubes (Beckman Instruments, Mijdrecht, The Netherlands). The virus suspension was reinforced with an equal volume of OptiPrep (Nycomed Pharma As, Oslo, Norway). The sealed tubes were rotated (290 minutes, 10 RPM) at an angle of 80 degrees. The virus was separated by density centrifugation (3 hours at 71,000 RPM) on a Vti80 bull (Beckman Instruments). The fractions were collected (200 ul per fraction) and titrated by the presence of recombinant AAV. The positive fractions were pooled (+ 400-600 ul) and diluted in 15 ml of PBS (NPBI) and then concentrated with Centriplus 100 and Centricon 100, respectively (Amicon, Capelle a / d Ijssel).

The concentrated fractions were titrated and found to have a titre of 8.5 x 109 infectious units per ml.

Table 1. Number of colonies after transfection of PER.C6 with E2A expression vectors Plasmid number of colonies established cell lines pcDNA3 «200 4/4 pcDNA3wtE2A 4 1/4 pcDNA3tsE2A «100 37/45 10 Table 2. Number of Colonies after transfection of 293 with E2A expression vectors Plasmid number of colonies pcDNA3 15 «100 pcDNA3wtE2A 2 pcDNA3tsE2A 25 Table 3. Infection of PER.C6 and PER.C6tsE2A c3-9 with Ad.dl802 twenty ÜNHMMÉMIÜ Table 4. Recombinant AAv adenovirus-free production in PER.C6 and PER.C6tsE2A.c5-9 Auxiliary Title function of rAAV ACV-ßgal (IU / ml) adenovirus 5 Plasmids PER.C6 PER.C6tsE2A.c5-9 pWE / Ad.? 5 '> 103 8 x 104 pWE / Ad.Ad-AflII-rITR 1.3 x 106 9.6 x 105 pE2a, pE4orf6, pVA 3.2 x 105 3.2 x 105 10 pE2a, pVA 3.2 x 105 4 x 104 pE2a, pE4orf6 2 x 104 6.4 x 105 pEorfd, pVA 10 1 x 104 pE2a 2.5 x 104 6.4 x 103 PVA 0 1.3 x 103 pEorf6 0 1.3 x 103 5 without Ad-DNA 10 1.3 x 103 Table 5. Transfection of PER.C6 cells in suspension.

Exp. DNA reagent (ug) DNA / lipid% Activity of noa Transfection (ug / ul) Blue ß-gal 0 ugrams / 106 cells DMRIE-CMR 4 1: 5 LO 1.52 0 0.0 -? jf l *******. .or-.

Table 5. Transfection of PER.C6 cells in suspension. (Continued) Exp. DNA reagent (ug) DNA / lipid% Activity of noa Transfection (ug / iul) Blue ß-gal ugrams / 106 cells 2 DMRIE-CMR 4 1: 5 20 3.8 - - 0 0.0 3 DMRIE-CMR 20 1:10 ND 0.53 - - ND 0.0 4 DMRIE-CMR 20 1:20 ND 0.10 FuGENEMR 6 20 1:20 ND 0.34 ND 0.0 Experiment 1 was carried out on 6-well discs in stationary cultures. Experiment 2 was carried out in 6 well disks in shaking cultures (100 RPM). During transfection the cultures were incubated without agitation. Experiments 3 and 4 were carried out in Erlenmeyer cultures under continuous agitation (100 RPM).

Table 6. Transfection of PER.C6 cells in suspension. DNA reagent PPZ% of cells Activity of Transfection (ug) (ug) blue v-β-gal viable cells (μg / 106 cells) PPZ 4 4 4 4 0 0 9 9 xx 110035 0.02 4 20 0 9 x 105 0.02 4 40 0 7 x 105 0.02 4 80 < 0. 1 8 x 105 0.02 4 160 < 0. 1 3 x 105 0.02 4 320 5 1.6 x 104 0.50 4 640 - < 103 4 1280 _ < 103 The number of viable cells, determined by trypan blue exclusion, two days after transfection.

Table 7. Transfection of PER.C6 cells in suspension. Reagent of .DN PPZ% of cells Transfection Activity ug) (ug) ß-gal viable cells blue-gal uGR / 106 cells) PPZ 4 160 5 4 x 105 ND 4 200 1 9 x 105 ND 4 240 - < 103 ND 4 280 _ < 103 ND | _ ^ u ^ | Hwdta Table 7. Transfection of PER.C6 cells in suspension. (continued) DNA Reagent PPZ% of elulas Activity of Transfection (ug) (ug) ules blesVml ß-gal blue cells / 106 cells 4 320 < 103 ND 4 360 < 103 ND 4 400 < 103 ND 4 440 < 103 ND 10 The number of viable cells, determined by trypan blue exclusion, two days after transfection.

REFERENCES 15 Alien JM, Debelak DJ, Reynolds TC, Miller AD (1997) Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production. J-Virol 71: 6816- 22 issn: 0022-538x 20 Berns K (1996) Parvoviridae: The viruses and their replication. In: Fields Virology, Lippincott - Raven, New York Berns K, Bergoin M, Bloom M, Lederman M, Muzyczka N, Siegl G, Tal J, Tattersall P (1994) Parvoviridae. VIth Report of the international Committee on taxonomy of viruses. In: Virus taxonomy, Archives of Viology suppl. 10. Berns PC, Bohensky R (1987) Adeno-associated virus: an update. Adv. Virus Res. 32: 243-306 5 Berns KI (1990a) Parvoviridae and their replication. In: Virology, Raven Presa, New York Berns KI (1990b) Parvovirus replication. Microbiol, Rev. 54: 316-329 Crankcase (1990) Adeno-associated virus helper 10 functions In: CRC handbook of parvoviruses, CRO Press, Inc., Boca Ratón Chejanovsky N, Carter BJ (1989) Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effets on viral DNA replication. Virology 173: 120-128 15 Clark K, Sierra T, Johnson P (1997) Recombinant adeno-associated virus vectors mediate long-term transgene expression in muscle. Hum Gene Ther 8: 659-669 Conway JE, Zolotukhin St Muzyczka N, Hayward GS, Byrne BJ (1997) Recombinant adeno-associated virus type 2 20 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap. J-Virol 71: 6780-9 issn: 0022-538x Ensinger M, Ginsberg H (1972) Selection and preliminary charactetization of temperature-sensitive mutants 25 of type 5 adenovirus. J Virol 10: 328-339 *. jiaAafa && ümt ^^ ^ ^ ^^ Ferrari F, Xiao x, McCarty D, Samulski R (1997) New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors. Nat Med 3: 1295-1297 Ferrari FK, Samulski T, Shenk T, Samulski RJ (1996) 5 Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J-Virol 70: 3227-34 Flotte TR, Afione SA, Conrad C, McGrath SA, Solow R, Oka H, Zeitlin PL, Guggino WB, Carter BJ (1993) Stable in 10 vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector, Proc Nati Acad Sci USA 90: 10613-1-617 Graham FL, Smiley J, Russell WC, Nairn R (1977a) Characteristics of a human ceil line transformed by DNA from 15 adenovirus type 5. J Gen Virol 36: 59-72 Graham FL, Smiley J, Russell WC, Naiva R (1977b) Characteristics of a human cell line transtormed by DNA from adenovirus type 5. J Gen Virol 36: 59-72 Hehir K, Armentano D, Cardoza L , Choquette T, 20 Berthelette P, White G, Couture L, Everton M, Keegan J, Martin J, Pratt D, Smith M, Smith A, Wadsworth S (1996) Molecular characterization of replication of variants of adenovirus vectors and genome modifications to prevent their occurence. J Virol 70: 8459-8467 m-'nfM "l! *" ~ i- ~ Herzog, R, Hagstrom J, Kung S, Tai S, Wilson J, Fischer K, High K (1997) Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Nati Acad Sci USA 94: 5804-5809 Hinds P, Finlay C, Quartin R, Baker S, Fearon E, Vogeistein B, Levine A (1990) Mutant p53 DNA clones from human colon carcinomas coorperate with ras in transforming primary mouse cellS: a comparison of the "hot spot" mutant phenotypes. Cell growth differ. 1: 571-580 Hong G, Ward P, Berns KI (1992) in vi tro replication of adeno-associated virus DNA. Proc Nati Acad Sci USA 89: 4673-4677 Hong G, Ward P, Berns KI (1994) Intermediates of adeno-associated virus DNA replication in vi tro. J Virol 68: 2011-2015 Imler J, Chartier C, Dreyer D, Dieterle A, Sainte-Marie M, Faure T, Pavirani A, Methali M (1996) Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of El-deleted adenovirus vectors, Gene Ther 3: 75-84 Kotin RM, Siniscalco M, Samulski RJ, Zhu X, Hunter L, Laughlin S, Muzyczka N, Rocchi M, Berns KI (1990) Site-specific integration by adeno-associated virus. Proc Nati Acad Sci USA 87: 2211-2215 Li J, Samulski R, Xiao X (1997) Role for highly regulated gene expression in adeno-associated virus vector production. J Virol 71: 5236-5243 Lochmuller H, Jani A, Huard J, Prescott S, Simoneau 5 M, Massie B, Karpati G, Acsadi G (1994) Emergence of early region 1-containing replication-competent adenovirus in stocks of replication- defective adenovirus recombinants (DEI + DE3) during multiple passages in 293 cells. Hum Gene Ther 5: 1485-1492 10 Louis N, Evelegh C, Graham F (1997) Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. J Virol 233: 423-429 Lusby E, Fife KH, Berns Kl (1980) Nucleotide 15 sequence of the inverted terminal repeat in adeno-associated virus DNA. J Virol 34: 402-409 Martinez C, Luna da S, Peleaz E, Lopez-Turiso A, Valcarcel J, Haro de O, Ortin J (1989) Permanent cell lines that show temperature-dependent expression of adenovirus 20 virus associated RNA . J Virol 63; 5445-5450 McCarty DM, Christensen M, Muzyczka N (1991) Sequences required for coordinate induction of adeno-associated virus pl9 and p40 promoters by Kep protein. J- Virol 65: 2936-45 issn: 0022-538x ? ti? Ttri] ii ????? írft? ira "" g ~ M --'- "'- - Monahan P, Samulski R, Tazelaar J, Xiao C, Nichols T, Bellinger D. Read M, Walsh C (1998) Direct intramuscular injection with recombinant AAV-vectors results in the expression of a hemophilia dog model Gene Ther 5: 40-49 5 Ni T, Zhou X, McCarty D, Zolotukhin I, Muzyczka N (1994) In vitro replication of adeno-associated virus DNA. J Virol 68: 1128-1138 Reich N, Sarnow P, Duprey E, Levine A (1983) Monoclonal antibodies which recognize native and denatured 10 forms of the adenovirus DNA-binding protein. Virology 128: 480-484 Rice SA, Klessig DF (1985) Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein. J Virol 56: 767-778 15 Ruffing M, Heid H, Kleinschmidt JA (1994) Mutations in the carboxy terminus of adeno-associated virus 2 capaid proteins aftect viral infectivity: lack of an RGD integrin-binding motif. J-Gen-Virol 75: 3385-92 Rutledge E, Halbert C, Russell D (1998) Infectious 20 clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV-2. J Virol 72: 309-319 Samulski RJ (1993) Adeno-ansociated virus. Integration at a specific chromosomal locus. Curr Opin Gen Dev 3: 74-80 Samulski RJ, Chang L, Shenk T (1989) Helper-free stocks ot recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 63: 3822-3828 5 Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA (1991) Targeted integration of adeno-associated virus (MV) into human chromosome 19. EMBO J. 10: 3941-3950 Smith a, Kotin a (1998) The Rep52 gene product of 10 adeno-assoctated virus is a DNA helicase with 3'-to 5 'polarity. J Virol 72: 4874-4881 Snyder R, Miao O, Patijn J, Spratt S, Danos 0, Nagy D, Gown A, Winther B, Meuse L, Cohen L, Thompson A, Kay M (1997a) Persistent and therapeutic concentration of human 15 factor IX in mice after hempatic gene transfer of recombinant AAV vectors, Nat. Genetics 16: 270-276 Snyder RO, Spratt SK; Lagarde C, Bohl-D, Kaspar B, Sloan B, Cohen LK, Danos O (1997b) Efficient and stable adeno-associated virus-mediated transduction in the skeletal 20 muscle of adult immunocompetent mice. Hum-Gene-Ther 8: 1891-900 issn: 1043-0342 Srivastava A, Lusby E, Berns K (1983) Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol 45: 555-564 rti '-jaft ^ M' Vincent AJPE, Esandi Mdc, Someren GDv, Noteboom JL, C.J.J A, Vecht 0, Smitt PAES, Bekkum DWv, Valerio D, Hoogerbrugge PM, Bout A (1996) Treatment of Lepto-meningeal metastasis in a rat model using a recombinant adenovirus containing the HSVtk gene. J. Neurosurg. 85: 648-654 Vincent KA, Piraino ST, Wadsworth SC (1997) Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products. J-Virol 71: 1897-905 issn: 0022-538x Vliet van der P, Levine A, Ensinger M, Ginsberg H (1975) Thermolabile DNA binding proteins from infected cells with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J Virol-15: 348-354 Ward P, Dean F, O'Donnell Mr Berns K. (1998) Role of the adenovirus DNA-binding protein in vi tro adeno-associated virus DNA replication. J Virol 72: 420-427 Xiao X, Li J, Samulski R (1998a) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72: 2224-2232 Xiao X, Li J, Samulski RJ (1998b) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus, J-Viroí 72: 2224-32 issn; 0022-538x Yacobson E, Hrynko T, Peak M, Winocour E (1989) Replication of adeno-associated virus in cells irradiated with UV light at 254 nm J Virol 63: 1023-1030 Yalkinoglu A, Heilbronn R, Burkle A, Sahiehofer J , zur Hausen H (1988) DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res 48: 3123-3125 Zhou 3, Murphy J, Escobedo J, Dwarki V (1998) Adenoassociated virus-mediated delivery of erythropoietin leads elastomérica rising from hematocrit in nonhuman primates. Gene ther 5: 655-670 Zhou x, Muzyczka N (1998) In vitro packaging of adenos associated virus DNA. J Virol 72: 3241-3247 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. k á ^ hlwKiM4a

Claims (1)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. 5 1. A cell for packaging adeno-associated viruses (AAV), characterized in that it has been provided with nucleic acid that codes for at least one genetic product that provides auxiliary function for the AAV that allows generating Recombinant AAV without auxiliary virus production 10 recombinant. 2. The cell in accordance with the claim 1, characterized in that the nucleic acid codes for an adenovirus gene product. 3. The cell according to claim 15 2, characterized in that the genetic product comprises the adenovirus or a functional fragment thereof. . The cell according to the claim 2, characterized in that the genetic product comprises E2A of adenovirus or a functional fragment thereof, where the E2A 20 is preferably derived from the tsl25 adenovirus. 5. The cell according to any of claims 1 to 4, characterized in that it has also been provided with at least one additional nucleic acid encoding the additional auxiliary function required. Mfiaat * < ií¡? m? 6. The cell according to claim 5, characterized in that the genetic product encodes for the Adenovirus El or a functional fragment thereof and at least one additional nucleic acid encodes for the E2A of 5 adenovirus or a functional fragment thereof, wherein the E2A is preferably derived from the ts25 adenovirus. 7. The cell in accordance with the claim 6, characterized in that the nucleic acid encoding the E4orf6 of adenovirus or a functional fragment thereof. 10. The cell according to any of claims 1 to 7, characterized in that it has been derived from a human embryonic retinoblastic cell. 9. The cell according to claim 8, characterized in that a PER.C6 cell that has been 15 deposited under accession number 96022940 ECACC at the Center for Applied Microbiology Research (CAMR) or a cell derived from it. 10. The cell according to any of claims 1 to 9, characterized in that it comprises the 20 cosmid pWE / Ad.AfHl-rlTR (ECACC deposit p97082116 in the CAMR). 11. The cell according to any of claims 1 to 10, characterized in that it comprises the cosmid pWE / Ad.AflII-rITR? E2A. 12. The cell according to any of claims 1 to 11, characterized in that it comprises the pcDNA3wtE2A plasmid. 13. A cell culture, characterized in that it comprises a cell according to any of claims 1 to 12. 14. The cell culture according to claim 13, characterized in that it comprises means without any human or animal constituent. 15. The cell culture according to claim 13 or 14, characterized in that it comprises a cell culture in suspension or another large-scale culture. 16. The use of a cell according to any of claims 1 to 12 or a culture Cell according to any of claims 13 to 15 to produce recombinant adeno-associated viruses. ^ jg ^ t? Ék